1. Field of the Invention
The present invention relates to DNA oligonucleotides that bind specifically to transcriptional regulatory proteins (TRPs) and the use of such oligonucleotide sequences in (a) the detection and/or isolation of TRPs, (b) analysis of TRP induction by cytokines and (c) analysis of TRP action.
2. Description of the Background Art
Cell surface receptors for extracellular polypeptide ligands are widely expressed on most mammalian cells and serve to transduce proliferative and differentiation signals to the cell's interior. Multistep signaling pathways lead from the binding of growth factor receptors, for example, the epidermal growth factor receptor (EGFR) to subsequent intracellular processes (J. Schlessinger et al., Neuron 9, 383 (1993)). Many growth factor receptors, like EGFR, possess intrinsic tyrosine kinase activities, which, after ligand activation, autophosphorylate the receptor cytoplasmic tail and phosphorylate other cellular substrates (A. Ullrich et al., Cell 61, 203 (1990)).
A common response for receptor and non-receptor signaling pathways involves the activation of Ras (L. S. Mulcahy et al., Nature 313, 241 (1985)). Autophosphorylated EGFR serves as a docking site for SH2 domain-containing proteins (C. A. Koch et al., Science 252, 668 (1991), which lead to Ras activation (E. J. Lowenstein et al., Cell 70, 431 (1992); N. Li et al., Nature 363, 85 (1993; N. W. Gale et al., Nature 363, 88 (1993); M. Rozakis-Adcock et al., Nature 363, 83 (1993); S. E. Egan et al., Nature 363, 45 (1993); M. Rozakis-Adcock et al., Nature 360, 689 (1992); K. Baltensperger et al., Science 260, 1950 (1993); J. P. Olivier et al. , Cell 73, 179 (1993)). Activated Ras then initiates a cascade of serine/threonine phosphorylations through Raf and MAP kinases (S. A. Moodie et al., Science 260, 1658 (1993); C. A. Lange-Carter et al., Science 260, 315 (1993)) leading to phosphorylation of nuclear proteins (C. S. Hill et al., Cell 73, 395 (1993); H. Gille et al., Nature 358, 414 (1992); R. H. Chen et al., Mol. Cell. Biol. 12, 915 (1992)).
It is widely speculated that the biological consequences of receptor tyrosine kinase activation depends on changes in gene expression. However, it has been difficult to document a direct connection between the cytoplasmic phosphorylation events stimulated by receptor tyrosine kinase activation and modulation of nuclear transcription factors. In contrast, recent studies of signals generated by interferons (IFNs) have revealed an apparently simpler path to the nucleus (D. E. Levy et al., New Biologist 2, 923 (1990)). Though lacking intrinsic catalytic domains (G. Uze et al., Cell 60, 225 (1990)), IFN.alpha. receptors stimulate tyrosine phosphorylation of a family of proteins that serve as DNA-binding and transcriptional-activating factors (C. Schindler et al., Science 257, 809 (1992); K. Shuai et al., Science 258, 1808 (1992); M. J. Gutch et al., Proc. Natl. Acad. Sci. USA 89, 11411 (1992)). These proteins, generically termed "signal transducers and activators of transcription (STATs)," normally sequestered in the cytoplasm (D. E. Levy et al., Genes Dev. 2, 1362 (1989); D. S. Kessler et al., Genes Dev. 4, 1753 (1990)), upon tyrosine phosphorylation, assemble into a multimeric complex, translocate to the nucleus, and bind cis-acting enhancer elements in the appropriate regulatory regions. One example of a STAT is ISGF3 which binds to regulatory regions of IFN-stimulated genes. IFN.gamma. stimulates phosphorylation of the 91 kDa protein subunit of ISGF3 (termed p91), leading to its appearance in the nucleus and its binding to a distinct genetic element, activating IFN.gamma.-responsive genes (K. Shuai et al., supra).
When mouse 3T3 fibroblasts are stimulated by v-sis (platelet-derived growth factor or PDGF), a nuclear factor, termed sis-inducible factor (SIF), binds to a DNA sequence which is a regulatory element in the c-fos promoter (T. E. Hayes et al., Proc. Natl. Acad. Sci. USA 84, 1272 (1987); B. J. Wagner et al., EMBO J. 9, 4477 (1990)). The rapid, transient stimulation of this DNA-binding activity led the present inventor and his colleagues to investigate its possible relationship to ISGF3 activation. This, led the present inventor to the discovery of the subject matter of the present invention, including (1) novel DNA oligonucleotide sequences to which such TRPs bind and (2) uses for these oligonucleotides.